A reduction-secretion system contributes to roxarsone (V) degradation and efflux in Brevundimonas sp. M20

A reduction-secretion system contributes to roxarsone (V) degradation and efflux in Brevundimonas sp. 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M20 Xuehui Zonga, Minghui Yu, Jiahui Wang, Congcong Li, Bing Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5363972/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Jan, 2025 Read the published version in BMC Microbiology → Version 1 posted 4 You are reading this latest preprint version Abstract Roxarsone (V) (Rox(V)) is an organoarsenical compound that poses significant risks to aquatic ecosystems and contributes to various diseases through its conversion into mobile inorganic and more toxic arsenic. Reducing trivalent 3-amino-4-hydroxyphenylarsonic acid (HAPA(III)) offers a competitive advantage; however, it leads to localized arsenic contamination, which can disrupt the soil microbiome and impede plant growth. Three genes, BsntrA , arsC 2, and BsexpA , encoding nitroreductase, arsenate reductase, and MFS transporter, were identified in a Rox(V) resistant strain Brevundimonas sp. M20. Then, a three-step approach, including nitroreduction, As (V) reduction, and HAPA(III) secretion, which is responsible for Roxarsone(V) resistance, was confirmed. Moreover, the flavonoid compound baicalin occupies the HAPA (III) delivery space and grabs the R127 residues by stronger interaction and steric hindrance to prevent HAPA (III) transported by BsexpA to the extracellular. These results demonstrate a new Rox(V) reduction pathway, providing a potential efflux pump inhibitor to trap higher toxins. Roxarsone (V) Nitroreductase Arsenate reductase MFS efflux pump Baicalin Efflux pump inhibitor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Highlights (1) Rox(V) is reduced and secreted by a three-step approach in sp. M20 ; (2) Rox(V) is reduced to HAPA(V) and HAPA(III) progressively by nitroreductase BsntrA and arsenate reductase ArsC2; (3) HAPA(III) is pumped out by MFS transporter BsexpA; (4) Flavonoid compound baicalin is a BsexpA inhibitor. Introduction Arsenic is one of the most prevalent water and environmental toxins [ 1 ] and ranks first on the US Priority List of Hazardous Substances [ 1 ]. The International Agency for Research on Cancer (IARC) has designated it as a Group 1 human carcinogen. Widely used for decades in poultry and pigs as feed additives to reduce coccidiosis infections and enhance growth, Roxarsone (3-nitro-4-hydroxybenzenearsenate or Rox(V)) and related synthetic aromatic arsenic compounds are organoarsenical compounds [ 2 ]. Unmetabolized roxarsone in animals is eventually excreted with manure and then converted into more poisonous and mobile inorganic arsenic (As), posing significant risks to aquatic ecosystems and water security [ 3 ]. Arsenical aromatic growth promoters are no longer permissible in the European Union or the United States, and they were recently prohibited in China. Although roxarsone remains a supplement in animal feed in developing countries [ 2 , 4 ]. Rox(V) demonstrates low toxicity; however, its application as fertilizer or during composting may lead to the formation of more toxic metabolites. The biodegradation of roxarsone in chicken feces and sewage sludge results in the formation of 3-amino-4-hydroxyphenylarsonic acid (HAPA-V) and arsenate has been previously documented [ 5 , 6 ]. In an oxic environment, a typical process in the microbial biotransformation of pentavalent Rox(V) involves the reduction of the nitro group, resulting in the formation of the more stable amine HAPA(V) [ 2 , 7 , 8 ]. This compound is ultimately degraded into inorganic As(V), As(III), and trivalent HAPA(III), Rox(III) [ 9 ]. FAD-NADPH-dependent nitroreductase, MdaB, from S. meliloti Rm1021 catalyzes the nitroreduction of Rox(V) in aerobic or anaerobic conditions [ 10 ]. The FMN-NADPH-dependent nitroreduction of Rox(III) to HAPA(III) is catalyzed by PpnfnB, a 6,7-dihydropteridine reductase from P. putida KT2440 (ΔΔars) [ 2 ]. Many diverse organisms persist in arsenic-contaminated environments, primarily due to the role of arsenite transporters despite the presence of arsenate reductase. During this phase, arsenite is either expelled from the cell's interior or preserved in vacuoles through an energy-dependent reaction facilitated by an arsenite transporter [ 11 ]. Plant-derived efflux pump inhibitors can inhibit these pumps, thereby enhancing the efficacy of antimicrobial agents [ 12 ]. Baicalin belongs to flavonoid compounds [ 13 ], which can inhibit multidrug resistance (MDR) pumps in bacteria [ 14 ]. Luteolin[ 15 ], 5'-Methoxyhydnocarpin-D[ 16 ] and 4′, 6′ -dihydroxy-3′, 5′-dimethyl-2′ -methoxychalcon [ 14 ] are proved to act as EPI by more vital interaction and steric hindrance. As a result, the microbial degradation of organoarsenicals results in localized arsenic contamination, which might modify the soil microbiota and impede plant growth [ 10 ]. We previously identified an arsenite-resistant strain, Brevundimonas sp. M20 with an arsHRNBC cluster was responsible for inorganic arsenic resistance [ 17 ]. In this study, we demonstrate that the Brevundimonas sp. M20 also contains a reduction-secretion system, which is composed of BsntrA (a putative FMN-dependent nitroreductase), ArsC2 (an arsenate reductase) and BsexpA (an MFS efflux pump) to provide a reduction to Rox(V). These three proteins are responsible for nitro reduction, AS(V) reduction and HAPA(III) secretion. Baicalin restored the effectiveness of Rox(V) as an EPI. These results demonstrate a new Rox(V) reduction pathway, providing a potential efflux pump inhibitor to trap higher toxins. Materials and Methods Bacterial strains and growth conditions Table S1 contains a list of the primary bacterial strains and plasmids used in this investigation. The strain of Brevundimonas sp . M20 was cultured at 28°C on LB agar or broth. Luria-Bertani (LB) broth or LB agar (0.5% yeast extract, 1% tryptone, 1% sodium chloride, 2% agar) treated with ampicillin (100 µg/ml) were the cultures used to grow Escherichia coli DH5α for genetic modification. Bioinformatics studies The Protein Basic Local Alignment Search Tool (BLASTP) was employed to identify homologues in databases of nonredundant protein sequences. Clustal Omega [ 18 ] and ESPript [ 19 ] were used for the multi-sequence comparison. BsNtrA, ArsC2, and BsExpA homologous models were run using Discovery Studio (v.2.0) software [ 20 ]. Molecular docking of BsNtrA with Rox(V), ArsC2 with HAPA(V), and BsExpA with HAPA(III) was conducted using the CDOCKER protocol in Discovery Studio 2.0. Cloning of the reduction and secretion genes into E. coli Genes were cloned and expressed as previously described [ 21 ]. The promoter sequence fragment of the ampicillin resistance gene (Pampr) from pMD-18T was amplified using Kpn I at the 5’ end with primers Pamp-F3 and Pamp-R3. Using the primers BsNtrA-F3 and BsNtrA-R3, the complete target gene of BsNtrA (designated as RS09665) was amplified from strain M20 by PCR. After fusing Pamp r and BsNtrA to produce p-BsNtrA fragments, they were digested using Xba I and Kpn I and then added to the pMD18-T vector. To assess the conversion from Rox(V) to HAPA(V), the resultant pMD-BsNtrA recombinant plasmid was converted into Escherichia coli DH5α and given the designation E. coli EcNtrA. Using ArsC2-F3 and BsExpA-R3 primers, arsC2 and BsExpA (designated as RS14070 and RS14075) with Xba I at 3' of BsExpA were amplified by PCR. A BsNAE fragment was obtained by fusing these segments. Following its digestion with Xba I and Kpn I, the BsNAE fragment was introduced into the pMD18-T vector. E. coli EcNAE3 was obtained by transforming the resultant pMD-BsNAE recombinant plasmid into Escherichia coli DH5α. The controls were E. coli that included BsNtrA ( E. coli EcN1), arsC2 (E. coli EcC1), and BsExpA ( E. coli EcE1), respectively. Combination of two genes, BsNtrA + arsC 2 ( E. coli EcNC), BsNtrA + BsExpA ( E. coli EcNE), and arsC 2 + BsExpA ( E. coli EcCE) were constructed by using the same process. As controls, DH5α and DH5α carrying the pMD18-T vector (DT) were employed (Table S2). Minimum Inhibitory Concentration (MIC) calculation The MIC test for Rox(V) isolates was performed on LB plates. M20 was initially activated on the LB plate at 28°C until a single colony formed. After that, individual colonies were moved into LB medium and cultured overnight at 28°C and 180 rpm. After adjusting the strain culture's OD 600nm to 0.1, it was diluted 100 times with an LB medium. Lastly, a 0.25 increment of the diluted culture was streaked on LB plates that contained 0.25–6 mM of filtered Rox(V). For three days, the plates were infected at 28°C. The same procedure was used to test E. Coli MICs. As controls, E. Coli DH5α and E. Coli DT (DH5α with pMD18-T) were employed. As previously mentioned, HAPA(III) was produced by chemically reducing Rox(V) following its reaction with BsNtrA and ArsC2 [ 22 ]. MIC tests were performed using the same procedure for HAPA(V) and HAPA(III) of various E. coli . At final concentrations of 8.0 mg/L, MICs of MB and CST in the presence of baicalin were employed as EPIs. Nitroreductase and arsenate reductase expression and purification Primers were used to amplify the BsNtrA and arsC2 genes by PCR (Table S2). Plasmid pMD18-NtrA and pMD18-arsC2 were obtained by cloning the PCR products into pMD18-T after they were purified using agarose gel electrophoresis. Plasmid pET-NtrA and pET-arsC2 were obtained by inserting the BsNtrA and arsC2 genes into the pET-15b vector digested with Nde I and Xho I, respectively, following sequence verification of the recombinant pMD18-T plasmids. The recombinant plasmids were subsequently cultured after being converted into Escherichia coli BL21 (DE3) cells and designated names such as E. coli DENA1 and DEAC3. When the optical density at 600 nm reached 0.6, 1.0 mM isopropyl-β-D-1-thiogalactopyranoside was administered to the cell to synthesize the His-tagged BsNtrA protein. For four to five hours, the culture was incubated at 28°C. As previously reported, the His-tagged BsNtrA was purified using a Ni-NTA-Sefinose Column (Sangon) and subsequently examined using 10% SDS-PAGE [ 23 ]. ArsC2 was purified by using the same process. Nitroreductase and arsenate reductase activity analysis The capacity of BsNtrA for reduction of Rox(V) (Sigma-Aldrich, MO, USA) was determined by Nitroreductase Activity Assay Kit (ShanghaiFushengIndustrialCo., Ltd., China) in vitro. Briefly, Rox(V) was incubated at 37°C in the presence or absence of 50µL (1µM) BsNtrA in a reaction solution (1 µM FMN/FAD and 500 µM NAD(P)H in 20 mM Tris/HCl, pH 8, and 100 mM NaCl. 100µL horseradish peroxidase (HRP) was added to the mixture and incubated at 37℃ for 15 min in the dark. The absorbance of the mixture was measured at 540nm wavelength. 3-amino-4-hydroxybenzenearsonic acid (AHA) was used as a control (CAS: 2163-77-16). The capacity of ArsC2 for reduction of HAPA(V) (Hubei Dahao Technology Co., Ltd., China) was determined by the Arsenate Reductase Activity Assay Kit (Shanghai Bohu Biological Technology Co., Ltd., China) in vitro. Briefly, 100µl (1µM) HAPA(V) was added into 50µl PBS buffer and incubated at 37℃ for 60 min after antibody addition. TMB substrate 100µl acted for 20 minutes at 37℃ in the dark. Difference values of absorbance (S) in the ArsC2 mixture and negative control (N) at 450nm (W1) and 630nm (W2) were measured. 1-Amino-2-hydroxybenzene (CAS: 95-55-6) was used as a control. Results BsNtrA and ArsC2 confer resistance to Rox(V) and HAPA(V) of M20 To investigate the Rox(V) resistance mechanism of the inorganic arsenic-resistant opportunistic pathogen Brevundimonas sp. M20 genes on the M20 genome were analyzed. Downstream ars cluster [ 17 ] that contributes to arsenite methylation, three Rox(V) resistance genes were identified (Figure S1 ). Genes of FKQ52_RS09665, FKQ52_RS14070 and FKQ52_RS14075, encoding nitroreductase, arsenate reductase and MFS transporter, were named BsNtrA , arsC 2 and BsExpA respectively (Fig. 1 A). The toxic trivalent amino aromatic derivative 4-hydroxy-3-aminophenylarsenite (HAPA(III)) was developed in Sinorhizobium meliloti by the sequential reduction of the arsenate group in Rox(V) after nitroreductase catalyzed the reduction of the nitro group to form an intermediate product 4-hydroxy-3-aminophenylarseate (HAPA(V)) [ 10 ]. Thus, resistance to Rox(V) and HAPA(V) of M20 was tested. M20 showed resistance to both Rox(V) and HAPA(V), with MIC of 1.25 mM and 1.50 mM, respectively (Fig. 1 B). Functions of BsNtrA, ArsC2 and BsExpA in M20 were analyzed by gene expression via pMD18-T. MICs of E. coli EcNAE3 (containing BsNtrA , arsC 2 and BsExpA ), EcN1(containing BsNtrA ), EcC1(containing arsC 2), EcE1 (containing BsExpA ), EcNC (containing BsNtrA and arsC 2), EcNE (containing BsNtrA and BsExpA ), and EcCE (containing arsC 2 and BsExpA ) to Rox(V) and HAPA(V) were tested. The MIC of Rox(V) against E. coli BsNAE3 was raised to 1.875 mM compared to DH5α and DT. When incubated in the presence of HAPA(V), E. coli BsNAE3 also emerged as a resistance, with a MIC of 1.75 mM. Thus, the expression of BsNtrA , arsC 2 and BsExpA in E. coli brought the Rox(V) and HAPA (V) resistance (Fig. 1 C). When any two genes of BsNtrA , arsC 2 and BsExpA were co-expressed in E. coli , MICs to Rox(V) and HAPA (V) showed differences (Fig. 1 C). For EcNC, with BsNtrA and arsC 2 co-expression, Rox(V) and HAPA (V) resistance increased slightly, compared to DH5α and DT. Meanwhile, MICs to Rox(V) and HAPA (V) were 0.75 mM and showed no significant differences. BsNtrA and BsExpA co-expression did not offer Rox(V) and HAPA (V) resistance, compared to DH5α and DT. Meanwhile, resistance to Rox(V) and HAPA (V) exhibited significant differences when arsC 2 and BsExpA were co-expressed. A slight increase of Rox(V) resistance (MIC = 0.25 mM) and a significant increase of HAPA (V) resistance (MIC = 3.75 mM) were observed. Single gene expression in E. coli showed no changes of MICs to Rox(V) and HAPA (V) in EcN1 and EcE1. MIC of Rox(V) and HAPA (V) toward EcC1, with arsC 2 expression, showed a similar increase when arsC 2 and BsExpA were co-expressed (Fig. 1 C). Thus, the significant rise of HAPA (V) resistance in E. coli DH5α was attributed to the arsC 2 expression. Considering the substantial increase in Rox(V) resistance in BsNtrA , arsC 2 and BsExpA co-expression strain EcNAE3 and protein functions, BsNtrA provided the catalysis of the nitro group in Rox(V). Accordingly, it was suggested that Rox(V) reduction in M20 is a two-step simultaneous decrease of the nitro group and arsenic atom, similar to that found in S. meliloti [ 10 ]. BsNtrA FMN interaction pocket The protein structure of BsNtrA was modeled using homology to investigate its function further. BsNtrA from Brevundimonas sp . M20 exhibited dimer formation, aligning with the homodimeric nature common to most nitroreductases [ 24 , 25 ]. To form a globular structure with expanded areas at both termini, two monomers of BsNtrA combine to form an α + β fold domain. With five β-strands and seven α-helices, each monomeric BsNtrA comprises three structural segments (Fig. 2 A). The FMN binding motif is characterized by a conserved sequence, "RRS-PDH-WEW-W×T/S" (Figure S2). Two FMN molecules could interact with both monomers in this homodimer by binding in the deep pockets of the dimeric interface. They have an opening to the surface and are attached to the pocket at the dimeric interface (Fig. 2 B). The coenzyme FMN's isoalloxazine ring is orientated with its isoalloxazine ring packed against the β3 backbone atoms. It extends into the deep pockets created by the other dimeric subunit's Trp131 residue, the α5-helix, and the α2–β1 loop from one of the monomers. P57-H57 and W148-T150 mediate the entrance of the isoalloxazine ring into the pocket. R26-S28 and R187-R188 mediate interactions with the ribityl moiety of FMN (Fig. 2 C). BsNtrA catalyzed the nitro group reduction of Rox(V) For Rox(V) nitroreduction, the open FMN cofactor-binding pocket of BsNtrA also provides a reaction zone. Residues H57, G58, K59 and FMN mediate interactions within 5.0 Å along the nitro of Rox(V). The imidazole group of His57, acylamino of Gly58 and amidogen of Lys 58 could interact with O6 of the nitro moiety in ROX(V) (Fig. 3 A). Meanwhile, the open reaction pocket facilitates the entry and exit of substrates and products (Fig. 3 B). A Nitroreductase Activity Assay Kit was used to assess the nitroreductase activity of BsNtrA in the presence of NAD(P)H and FMN. A combination of Rox(V) and BsNtrA was active. At 450 nm, however, the AHA and BsNtrA mixture displayed a decrease in absorbance (Fig. 3 C). Considering the difference between Rox(V) and AHA, a nitro group, this result confirmed that BsNtrA can reduce nitro-containing Rox(V). ArsC2 has HAPA(V) reduction catalytic activity Inorganic arsenate must first be reduced to arsenite by arsenate reductase (ArsC) before it is extruded [ 26 ]. arsNRBC operon was identified in previous research in the M20 chromosome (unpublished). Another arsenate reductase encoded by RS14070 and named ArsC2 in the M20 chromosome was determined. As an organic arsenic compound, Rox(V) is not a suitable substrate for ArsC (Fig. 4 A). Surprisingly, HAPA(V) reduced activated the arsenate reductase enzyme activity of ArsC2 (Fig. 4 A). As an analogue of HAPA(V), 1-Amino-2-hydroxybenzene (AHB) could not activate the arsenate enzyme activity of ArsC2 because of the AsO 4 3− deficiency (Fig. 4 A). Thus, these results showed a reduction of HAPA(V) to HAPA(III) mediated by ArsC2. To investigate the detoxification mechanism, we analyzed the details of ArsC2 and its catalytic function. It indicated that ArsC2 mutated into a unique protein in M20. As reductases, essential redox-active cysteines are catalytic residues in ArsC, but the cysteine numbers vary. ArsC contains three cysteines (Cys10, Cys82 and Cys89) in Staphylococcus aureus [ 27 ] and Bacillus thuringiensis [ 28 ], two cysteines in E. coli (Cys12 and Cys106) [ 29 ] and two cysteines in M20 (Cys11 and Cys105). Unlike these arsenate reductases, ArsC2 only contains one cysteine (Cys11, equal to Cys12 in E. coli ), the catalytic residue [ 30 ]. ArsC2 maintained the HX3CX3R type catalytic sequence motif, which is necessary for arsenic detoxification activity in the traditional arsenate-reductase family, in addition to Cys11, His7, and Arg15 [ 31 ] (Fig S3). This conservative motif provided a catalytic ability to pentavalent arsenic. Like ArsC in E. coli (PDB: 1I9D), ArsC2 of M20 comprised four-stranded β-sheets and seven α-helices. The difference between them is the composition and structure of random coils (RC). Catalytic residue at the end of α1 was sounded by three random coils (RC1-3) to form an active site loop, and compared to 1I9D, RC1-3 of ArsC2 provided a larger substrate binding space (Fig. 4 B). The amino hydroxybenzene tail of HAPA(V) is embedded into this loop and stabilized by residues, such as Thr13, Val17, Arg93, Val103, Leu104, Ala105 and Arg106 (Fig. 4 C). Moreover, RC1-3 offered a higher hydrophobic environment-sounding active site, especially the mutation of Cys106 in 1I9D to Ala105 in ArsC2. This increased hydrophobic environment created a hydrophobic trap to enter HAPA's amino hydroxybenzene tail (V). Besides the hydrophobic trap, substrate binding space changed from a canyon-like type (11.477×9.826 Å) to a basin-like type (12.264 ×18.380 Å) (Fig. 4 D). This expanded area, combined with the hydrophobic trap, may be a reasons for substrate specificity changes from inorganic arsenate to organic arsenic. BsexpA has HAPA(III) efflux pump function Considering that expression of BsexpA alone in E. coli did not increase the MIC to Rox(V) and HAPA(V) (Fig. 1 C), we concluded that BsexpA is a HAPA(III) specific efflux pump and investigated the transport mechanism and cure for Rox(V) resistance. BsexpA is a Brevundimonas sp. specific MFS family transporter. Rosetta Design's homologous modeling revealed that the structure of BsexpA comprises two domains (the C domain and the N domain) and 12 TM helices (TMs 1–12). The interaction of BsexpA and HAPA(III) analysis indicated two substrate binding sites (SBS1 and SBS2) (Fig. 5 A). In SBS1, N32, A33, I36, and D37, residues bind mainly to HAPA(III) with carbon-hydrogen bond, Pi-alkyl and unfavorable acceptor-acceptor type acting forces. In SBS2, interacting residues moved to R127, V128, S354 and G358 with attractive charge and π bond forces (Fig. 5 A), which are weaker than SBS1. These two binding sites indicated a substrate delivery between them when conformations change outward-open and inward-open in typical MFS [ 32 ]. Potential flavonoid EPI baicalin (CAS: 21967-41-9) (Fig. 5 B) has been investigated previously [ 21 ]. The inhibition of baicalin towards BsexpA was then evaluated using molecular simulation. The baicalin binding site (BBS) was in the middle cavity between SBS1 and SBS2 (Fig. 5 C up). Nine residues, including HAPA(III) binding residue R127 in SBS1, interacted with baicalin by more solid bonds such as van der Waals, salt bridge, Pi-anion and carbon-hydrogen bond (Fig. 5 C down). This binding characteristic of baicalin with BsexpA provided two effects on HAPA (III) transport. Firstly, stronger interaction of BsExpA with baicalin than with HAPA(III) may make it easier to bind with baicalin. Secondly, once baicalin binds to BsExpA, BBS occupies the HAPA (III) delivery space from SBS1 to SBS2 by steric hindrance and grabs the R127 residues to prevent HAPA (III) binding to SBS2. Thus, these characteristics may prevent HAPA (III) transportation to the extracellular. Effects of baicalin on BsexpA were investigated. For M20 and EcE1, baicalin decreased MIC towards HAPA(III) to 8 mM). This result is consistent with a previous report that the MIC of baicalin to E. coli strains was 8.96 mM [ 33 ]. Discussion The contamination of organoarsenic is becoming increasingly prominent [ 34 ]. Roxarsone is an organoarsenical compound widely used for decades and is a significant threat to the eco-environment and human health [ 35 ]. It has been demonstrated that microbial communities are crucial to the worldwide biocycles of arsenic [ 36 , 37 ]. Brevundimonas sp. M20, isolated previously, is resistant to inorganic arsenic and Rox(V). arsHRNBC cluster in the M20 genome is responsible for inorganic arsenic resistance. This study demonstrates that an FMN-dependent nitroreductase BsntrA and an arsenate reductase ArsC2 provide the resistance to Rox(V). These two protein is responsible for the Rox(V) nitro reduction and AS(V) reduction, respectively. These findings reveal a novel mechanism that provides defense against environmental aromatic arsenicals. The reduction of nitro groups in nitroaromatic and nitro heterocyclic compounds to amino or hydroxylamino groups is mediated by the enzyme nitroreductase, which depends on FMN[ 37 , 38 ]. The nitroreductase family has been identified in eukaryotic, archaeal, and bacterial species [ 25 , 37 ]. Nitroreductases with broad substrate specificity can catalyze both natural and produced molecules. As a result, the biotechnological uses of nitroreductases in prodrug activation gene therapy for the treatment of cancer, bioremediation [ 39 ], and biocatalysis [ 40 ] make them extremely attractive [ 41 ]. PpNfnB from P. putida catalyzes FMN-NADPH-dependent nitroreduction of Rox(III) to less toxic HAPA(III) and confers resistance to Rox(V) [ 2 ]. MdaB from Sinorhizobium meliloti both reduces MAs(V) to MAs(III) and catalyzes sequential two-step reduction of nitro and arsenate groups in Rox(V), producing the highly toxic trivalent amino aromatic derivative HAPA(V) [ 10 ]. Crystal structures of nitroreductases, such as YdjA [ 24 ] and NfsA [ 42 ] in E. Coli , Frm2 [ 43 ] from Saccharomyces cerevisiae were elucidated. Nitroreductase of M20, BsNtrA, formed a dimer by seven α-helices and five β-strands, consistent with the homodimer characteristic of most nitroreductases [ 24 , 25 ]. The activity confirmed that BsNtrA can reduce nitro-containing Rox(V). This nitroreductase provides the nitro reduction of Rox(V). Thus, BsNtrA is a promising enzyme for bioremediation. Arsenate reductase (ArsC) facilitates the two-electron reduction of arsenate to arsenite, serving as the essential enzyme in the arsenate reduction process [ 44 ]. arsC is usually located in ars operons in various microorganisms [ 45 ]. ArsC contains three cysteines (Cys10, Cys82 and Cys89) in Staphylococcus aureus [ 27 ] and Bacillus thuringiensis [ 28 ], two cysteines in E. coli (Cys12 and Cys106) and two cysteines in M20 (Cys11 and Cys105). Cys12 was identified as the catalytic residue and is activated by a nearby positive charge, His-8, in E. coli ArsC [ 29 ]. However, Besides ArsC encoded by arsC gene in an ars operon, M20 contains a novel arsenate reductase encoding gene ( arsC 2). The organic arsenic compound HAPA(V), reduced from Rox(v), is not a suitable substrate for ArsC but activates the arsenate reductase enzyme activity of ArsC2. Thus, these results indicated a reduction of the HAPA(V) arsenate group mediated by ArsC2. Moreover, unlike two or more cysteines containing ArsCs, ArsC2 is a one-Cys containing (Cys11) arsenate reductase. The classical arsenate-reductase family's arsenic detoxification activity is contingent upon the remaining cysteines in the HX 3 CX 3 R-type catalytic sequence motif [ 31 ]. Compared to 1I9D from E. coli [ 29 ], mutations of residues that sound active site cavities not only provided a larger substrate binding space for HAPA(V) binding but also increased hydrophobic environment to create a hydrophobic trap to facilitate the higher hydrophobic substrate entry. These results may explain why ArsC2 could catalyze HAPA(V). Arsenite transporters enable various organisms to flourish in arsenic-contaminated environments despite the arsenate reductase. In this stage, an arsenite transporter catalyzes an energy-dependent process that pumps the arsenite out of the cell's interior or stores it in vacuoles [ 11 ]. To date, five types of arsenic transporters (ArsB [ 46 ], Acr3[ 47 ], ArsJ [ 48 ], ArsP [ 49 ], ArsK [ 50 ] and MSF1 [ 51 ]) have been found in various arsenic-resistant bacteria, and each confers resistance to different types of arsenic compounds [ 50 ]. ArsJ, ArsK and MSF1 belong to the major facilitator superfamily (MFS) and mediate the transport of various substrates [ 50 ]. Adjacent to the arsC 2 gene, a MFS transporter encoding gene BsExpA was identified. Gene expression showed that this gene contributes to resistance to Rox(V) and HAPA(V), partly only co-expressed with arsC2 . Thus, the function of BsExpA is related to the action of ArsC2. The deductive substrate is HAPA(V). The rapid emergence of antimicrobial resistance has become a serious clinical challenge. The therapeutic effectiveness of antibiotics is improved by identifying efficient EPIs that prevent bacteria from pumping antibiotics out of the cell [ 52 ]. Verapamil, reserpine, carbonyl cyanide-m-chlorophenylhydrazone, and β-naphthylamide have all been shown to be EPIs. However, most of these compounds' clinical uses were restricted by their toxicity, volatility, and low bioavailability; as a result, separating EPIs from natural products—such as flavonoids—has been a successful technique [ 15 ]. Luteolin [ 15 ], 5'-Methoxyhydnocarpin-D [ 16 ] and 4′, 6′ -dihydroxy-3′, 5′-dimethyl-2′ -methoxychalcon[ 14 ] were found to be EPIs in previous studies. As a flavonoid compound, baicalin inhibited MFS-type efflux pump BsExpA in this study. Unlike preventing the efflux pump from obtaining energy (such as luteolin towards MsrA[ 15 ]), baicalin binds to BsExpA by more solid bonds, compared to the bonds of substrate binding. Moreover, the baicalin binding site in the middle cavity between HAPA(III) binding sites grabbed R127, an essential HAPA(III) binding residue. Thus, these characteristics may offer baicalin a stronger interaction with BsExpA than HAPA(III) and a steric hindrance to prevent HAPA (III) transportation to the extracellular. The data presented herein indicates a contribution to Rox(V) resistance mediated by nitroreductase, arsenate reductase and MFS transporter in Brevundimonas sp . M20 (Fig. 6 ). This pathway reduces organic Rox(V) to HAPA(III) by a two-step reduction catalysis and pump-out. Firstly, nitroreductase BsNtrA catalyzes Rox(V) to HAPA(V) by reducing the nitro group into the amino group. Then, AsO 4 3− in HAPA(V) was reduced to AsO 3 3− by arsenate reductase ArsC2 to form HAPA(III). Thirdly, HAPA(III) was pumped out by MFS family transporter BsExpA. Elucidation of this novel Rox(V) resistance pathway adds an essential inspection to continue the arsenic resistance. Declarations Data Availability Statement The complete sequence of the Brevundimonas sp. M20 genome was deposited in GenBank under accession no. CP041243. Clinical trial number Not applicable. Acknowledgments We thank Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the language of a draft of this manuscript. Ethics approval and consent to participate Not applicable. Consent for publication The authors declare no competing interests. Funding This work was supported by the National College Students’ innovation and entrepreneurship training program [grant number S202410439004, 2024]. We thank Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the language of a draft of this manuscript. Author Contribution XHZ prepared the strain construction, the data analysis and wrote the manuscript. MHY the performance of bioinformatics analysis. JHW helped to analyze data. CCL helped to analyze data and revised the manuscript. 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Supplementary Files SupplementaryMaterialsBMCMicrobiology.docx Cite Share Download PDF Status: Published Journal Publication published 14 Jan, 2025 Read the published version in BMC Microbiology → Version 1 posted Editorial decision: Revision requested 12 Nov, 2024 Editor assigned by journal 06 Nov, 2024 Submission checks completed at journal 05 Nov, 2024 First submitted to journal 30 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5363972","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":376960751,"identity":"a45858c5-1d2c-4950-8f96-899a084013aa","order_by":0,"name":"Xuehui Zonga","email":"","orcid":"","institution":"Shandong University of Aeronautics","correspondingAuthor":false,"prefix":"","firstName":"Xuehui","middleName":"","lastName":"Zonga","suffix":""},{"id":376960752,"identity":"bcbb918f-0aeb-477e-8b24-f0acc5edf261","order_by":1,"name":"Minghui Yu","email":"","orcid":"","institution":"Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Minghui","middleName":"","lastName":"Yu","suffix":""},{"id":376960753,"identity":"3b85e8f4-2a58-457f-aff8-69cc7ecf7f4d","order_by":2,"name":"Jiahui Wang","email":"","orcid":"","institution":"Shandong First Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiahui","middleName":"","lastName":"Wang","suffix":""},{"id":376960754,"identity":"59ce8fd9-a5ca-43d8-b44f-8ab1a08e89e3","order_by":3,"name":"Congcong Li","email":"","orcid":"","institution":"Shandong Quancheng Test \u0026 Technology Limited Company","correspondingAuthor":false,"prefix":"","firstName":"Congcong","middleName":"","lastName":"Li","suffix":""},{"id":376960755,"identity":"6519b139-33b0-4dfe-9d54-edcfed65c578","order_by":4,"name":"Bing Wang","email":"","orcid":"","institution":"Shandong Quancheng Test \u0026 Technology Limited Company","correspondingAuthor":false,"prefix":"","firstName":"Bing","middleName":"","lastName":"Wang","suffix":""},{"id":376960756,"identity":"07813f84-b497-415b-8324-be91da5f654e","order_by":5,"name":"Yongan Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYHACxgcfKmx4+PkbiNfCbDjjTJqM5IwDxGthE+ZsO2xj0JBApHqDG8nPmBnOnOcxYDjA+OFjDlFa0sweF1Tc5jFnbmCWnLmNCC1mNxLMjWecuc1j2XCAjZmXOC3p36R5287xGBxIIFpLjhlQywEStNifeVMMDORkHskZB5uJ84tke/pGYFTa2fPzNx/88JEYLQwCCTAWYwMx6oGA/wCRCkfBKBgFo2DkAgCSpzrkuYabYAAAAABJRU5ErkJggg==","orcid":"","institution":"Shandong First Medical University","correspondingAuthor":true,"prefix":"","firstName":"Yongan","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-10-31 01:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5363972/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5363972/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12866-024-03740-4","type":"published","date":"2025-01-14T15:57:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69374180,"identity":"10333df5-a178-46e3-9bc9-a59b3cb2fd1d","added_by":"auto","created_at":"2024-11-19 16:49:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":78523,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBsNtrA and ArsC2 confer resistance to Rox(V) and HAPA(V) of M20.\u003c/strong\u003e (A) location of \u003cem\u003eBsNtrA, arsC2\u003c/em\u003e and \u003cem\u003eBsExpA\u003c/em\u003e. (C) MICs of strains with different genes expressed in DH5α to Rox(V) and HAPA (V). *MIC\u0026lt;0.25mM.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5363972/v1/e54ee25e1d53d41934e056e8.png"},{"id":69374182,"identity":"527366ec-b803-43a3-bfcd-500dd5f55b62","added_by":"auto","created_at":"2024-11-19 16:49:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":662017,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBsNtrA is a FMN depending protein. \u003c/strong\u003e(A) Structural overview of BsNtrA. (B) FMN binding pockets of BsNtrA. Two FMNs were represented by yellow-skeleton sticks and blue -skeleton sticks. (C) Interactions of BsNtrA with coenzyme FMN.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5363972/v1/34f79d7ff397cb83c74d5b20.png"},{"id":69374922,"identity":"4a5caba4-47a5-42c0-9b7b-6ddd6b69b0ed","added_by":"auto","created_at":"2024-11-19 16:57:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":459211,"visible":true,"origin":"","legend":"\u003cp\u003eBsNtrA catalyzed the nitro group reduction of Rox(V).\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Structural overview interactions of BsNtrA with coenzyme FMN and substrates Rox(V). (\u003cstrong\u003eB\u003c/strong\u003e) Open reaction pocket BsNtrA and substrate. (\u003cstrong\u003eC\u003c/strong\u003e) Nitroreductase activity of BsNtrA. Control: reaction mixture without BsNtrA; AHA: reaction mixture with AHA as substrate; Rox(V): reaction mixture with Rox(V) assubstrate.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5363972/v1/c8506910590ebfceca657474.png"},{"id":69374920,"identity":"f330489b-9d2d-4ac1-865d-16b035c358c1","added_by":"auto","created_at":"2024-11-19 16:57:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":828516,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArsC2 mediates the HAPA(V) reduction. \u003c/strong\u003e(A) Arsenate reductase activity test. (B) Structures of ArsC2 and its substrate binding space. (C) Interactions of HAPA(V) and ArsC2. (D) Hydrophobic trap in substrate binding space. 119D: ArsC from \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5363972/v1/7522b76c16858c6c160cb095.png"},{"id":69374921,"identity":"9db69e48-ff87-459f-a795-c0eda234cd32","added_by":"auto","created_at":"2024-11-19 16:57:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":761181,"visible":true,"origin":"","legend":"\u003cp\u003eBsexpA has HAPA(III) efflux pump function. (A) Two substrate binding sites of BsexpA. SBS1 and SBS2 are represented by carmine and green mesh. Binding residues of SBS1 and SBS2 are represented by orange and purple thin sticks. HAPA(III) in SBS1 and SBS2 are represented by carmine and green thick sticks. (B) Chemical structural formula of baicalin. (C) Baicalin occupies the HAPA (III) delivery approach and R127 residue to prevent HAPA (III) transportation. Baicalin binding site (BBS) is represented by blue mesh. Binding residues are represented by orange lines. (D) Baicalin decreased MIC of M20 and BsNAE3 strains, MICs of \u0026lt;0.25mM are defined as 0.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5363972/v1/200ac3f0ae9acb5b70ecb42c.png"},{"id":69374184,"identity":"4e53c3f7-b12a-4102-b349-a8a6bcf6aad6","added_by":"auto","created_at":"2024-11-19 16:49:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":222120,"visible":true,"origin":"","legend":"\u003cp\u003eContributions to Rox(V) resistance in \u003cem\u003eBrevundimonas\u003c/em\u003e sp. M20\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5363972/v1/19228612271b8de6645653a5.png"},{"id":74284489,"identity":"64eeb1b6-0663-41f8-bac1-6028556daef8","added_by":"auto","created_at":"2025-01-20 16:07:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4427170,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5363972/v1/65983f9b-eb6f-4322-84cc-ef8f83959f28.pdf"},{"id":69374185,"identity":"e6153910-557f-4a57-9b0a-b8448fd2a58f","added_by":"auto","created_at":"2024-11-19 16:49:13","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":926915,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialsBMCMicrobiology.docx","url":"https://assets-eu.researchsquare.com/files/rs-5363972/v1/0d72bcb55927b6431716435d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A reduction-secretion system contributes to roxarsone (V) degradation and efflux in Brevundimonas sp. M20","fulltext":[{"header":"Highlights","content":"\u003cp\u003e(1) Rox(V) is reduced and secreted by a three-step approach in sp. M20 ;\u003c/p\u003e\u003cp\u003e(2) Rox(V) is reduced to HAPA(V) and HAPA(III) progressively by nitroreductase BsntrA and arsenate reductase ArsC2;\u003c/p\u003e\u003cp\u003e(3) HAPA(III) is pumped out by MFS transporter BsexpA;\u003c/p\u003e\u003cp\u003e(4) Flavonoid compound baicalin is a BsexpA inhibitor.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eArsenic is one of the most prevalent water and environmental toxins [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and ranks first on the US Priority List of Hazardous Substances [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The International Agency for Research on Cancer (IARC) has designated it as a Group 1 human carcinogen. Widely used for decades in poultry and pigs as feed additives to reduce coccidiosis infections and enhance growth, Roxarsone (3-nitro-4-hydroxybenzenearsenate or Rox(V)) and related synthetic aromatic arsenic compounds are organoarsenical compounds [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Unmetabolized roxarsone in animals is eventually excreted with manure and then converted into more poisonous and mobile inorganic arsenic (As), posing significant risks to aquatic ecosystems and water security [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Arsenical aromatic growth promoters are no longer permissible in the European Union or the United States, and they were recently prohibited in China. Although roxarsone remains a supplement in animal feed in developing countries [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRox(V) demonstrates low toxicity; however, its application as fertilizer or during composting may lead to the formation of more toxic metabolites. The biodegradation of roxarsone in chicken feces and sewage sludge results in the formation of 3-amino-4-hydroxyphenylarsonic acid (HAPA-V) and arsenate has been previously documented [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In an oxic environment, a typical process in the microbial biotransformation of pentavalent Rox(V) involves the reduction of the nitro group, resulting in the formation of the more stable amine HAPA(V) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This compound is ultimately degraded into inorganic As(V), As(III), and trivalent HAPA(III), Rox(III) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. FAD-NADPH-dependent nitroreductase, MdaB, from \u003cem\u003eS. meliloti\u003c/em\u003e Rm1021 catalyzes the nitroreduction of Rox(V) in aerobic or anaerobic conditions [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The FMN-NADPH-dependent nitroreduction of Rox(III) to HAPA(III) is catalyzed by PpnfnB, a 6,7-dihydropteridine reductase from \u003cem\u003eP. putida\u003c/em\u003e KT2440 (ΔΔars) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany diverse organisms persist in arsenic-contaminated environments, primarily due to the role of arsenite transporters despite the presence of arsenate reductase. During this phase, arsenite is either expelled from the cell's interior or preserved in vacuoles through an energy-dependent reaction facilitated by an arsenite transporter [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Plant-derived efflux pump inhibitors can inhibit these pumps, thereby enhancing the efficacy of antimicrobial agents [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Baicalin belongs to flavonoid compounds [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], which can inhibit multidrug resistance (MDR) pumps in bacteria [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Luteolin[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], 5'-Methoxyhydnocarpin-D[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and 4\u0026prime;, 6\u0026prime; -dihydroxy-3\u0026prime;, 5\u0026prime;-dimethyl-2\u0026prime; -methoxychalcon [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] are proved to act as EPI by more vital interaction and steric hindrance. As a result, the microbial degradation of organoarsenicals results in localized arsenic contamination, which might modify the soil microbiota and impede plant growth [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe previously identified an arsenite-resistant strain, \u003cem\u003eBrevundimonas\u003c/em\u003e sp. M20 with an \u003cem\u003earsHRNBC\u003c/em\u003e cluster was responsible for inorganic arsenic resistance [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In this study, we demonstrate that the \u003cem\u003eBrevundimonas\u003c/em\u003e sp. M20 also contains a reduction-secretion system, which is composed of BsntrA (a putative FMN-dependent nitroreductase), ArsC2 (an arsenate reductase) and BsexpA (an MFS efflux pump) to provide a reduction to Rox(V). These three proteins are responsible for nitro reduction, AS(V) reduction and HAPA(III) secretion. Baicalin restored the effectiveness of Rox(V) as an EPI. These results demonstrate a new Rox(V) reduction pathway, providing a potential efflux pump inhibitor to trap higher toxins.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains and growth conditions\u003c/h2\u003e \u003cp\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e contains a list of the primary bacterial strains and plasmids used in this investigation. The strain of \u003cem\u003eBrevundimonas sp\u003c/em\u003e. M20 was cultured at 28\u0026deg;C on LB agar or broth. Luria-Bertani (LB) broth or LB agar (0.5% yeast extract, 1% tryptone, 1% sodium chloride, 2% agar) treated with ampicillin (100 \u0026micro;g/ml) were the cultures used to grow \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α for genetic modification.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBioinformatics studies\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe Protein Basic Local Alignment Search Tool (BLASTP) was employed to identify homologues in databases of nonredundant protein sequences. Clustal Omega [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and ESPript [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] were used for the multi-sequence comparison. BsNtrA, ArsC2, and BsExpA homologous models were run using Discovery Studio (v.2.0) software [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Molecular docking of BsNtrA with Rox(V), ArsC2 with HAPA(V), and BsExpA with HAPA(III) was conducted using the CDOCKER protocol in Discovery Studio 2.0.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCloning of the reduction and secretion genes into\u003c/b\u003e \u003cb\u003eE. coli\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenes were cloned and expressed as previously described [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The promoter sequence fragment of the ampicillin resistance gene (Pampr) from pMD-18T was amplified using \u003cem\u003eKpn\u003c/em\u003e I at the 5\u0026rsquo; end with primers Pamp-F3 and Pamp-R3. Using the primers BsNtrA-F3 and BsNtrA-R3, the complete target gene of \u003cem\u003eBsNtrA\u003c/em\u003e (designated as RS09665) was amplified from strain M20 by PCR. After fusing Pamp\u003csup\u003er\u003c/sup\u003e and \u003cem\u003eBsNtrA\u003c/em\u003e to produce p-BsNtrA fragments, they were digested using \u003cem\u003eXba\u003c/em\u003e I and \u003cem\u003eKpn\u003c/em\u003e I and then added to the pMD18-T vector. To assess the conversion from Rox(V) to HAPA(V), the resultant pMD-BsNtrA recombinant plasmid was converted into \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α and given the designation \u003cem\u003eE. coli\u003c/em\u003e EcNtrA.\u003c/p\u003e \u003cp\u003eUsing ArsC2-F3 and BsExpA-R3 primers, \u003cem\u003earsC2\u003c/em\u003e and \u003cem\u003eBsExpA\u003c/em\u003e (designated as RS14070 and RS14075) with \u003cem\u003eXba\u003c/em\u003e I at 3' of BsExpA were amplified by PCR. A BsNAE fragment was obtained by fusing these segments. Following its digestion with \u003cem\u003eXba\u003c/em\u003e I and \u003cem\u003eKpn\u003c/em\u003e I, the BsNAE fragment was introduced into the pMD18-T vector. \u003cem\u003eE. coli\u003c/em\u003e EcNAE3 was obtained by transforming the resultant pMD-BsNAE recombinant plasmid into \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α. The controls were \u003cem\u003eE. coli\u003c/em\u003e that included BsNtrA (\u003cem\u003eE. coli\u003c/em\u003e EcN1), \u003cem\u003earsC2\u003c/em\u003e (E. coli EcC1), and BsExpA (\u003cem\u003eE. coli\u003c/em\u003e EcE1), respectively. Combination of two genes, \u003cem\u003eBsNtrA\u0026thinsp;+\u0026thinsp;arsC\u003c/em\u003e2 (\u003cem\u003eE. coli\u003c/em\u003e EcNC), \u003cem\u003eBsNtrA\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eBsExpA\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e EcNE), and \u003cem\u003earsC\u003c/em\u003e2\u0026thinsp;+\u0026thinsp;\u003cem\u003eBsExpA\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e EcCE) were constructed by using the same process. As controls, DH5α and DH5α carrying the pMD18-T vector (DT) were employed (Table S2).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eMinimum Inhibitory Concentration (MIC) calculation\u003c/h3\u003e\n\u003cp\u003eThe MIC test for Rox(V) isolates was performed on LB plates. M20 was initially activated on the LB plate at 28\u0026deg;C until a single colony formed. After that, individual colonies were moved into LB medium and cultured overnight at 28\u0026deg;C and 180 rpm. After adjusting the strain culture's OD\u003csub\u003e600nm\u003c/sub\u003e to 0.1, it was diluted 100 times with an LB medium. Lastly, a 0.25 increment of the diluted culture was streaked on LB plates that contained 0.25\u0026ndash;6 mM of filtered Rox(V). For three days, the plates were infected at 28\u0026deg;C. The same procedure was used to test \u003cem\u003eE. Coli\u003c/em\u003e MICs. As controls, \u003cem\u003eE. Coli\u003c/em\u003e DH5α and \u003cem\u003eE. Coli\u003c/em\u003e DT (DH5α with pMD18-T) were employed. As previously mentioned, HAPA(III) was produced by chemically reducing Rox(V) following its reaction with BsNtrA and ArsC2 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. MIC tests were performed using the same procedure for HAPA(V) and HAPA(III) of various \u003cem\u003eE. coli\u003c/em\u003e. At final concentrations of 8.0 mg/L, MICs of MB and CST in the presence of baicalin were employed as EPIs.\u003c/p\u003e\n\u003ch3\u003eNitroreductase and arsenate reductase expression and purification\u003c/h3\u003e\n\u003cp\u003ePrimers were used to amplify the \u003cem\u003eBsNtrA\u003c/em\u003e and \u003cem\u003earsC2\u003c/em\u003e genes by PCR (Table S2). Plasmid pMD18-NtrA and pMD18-arsC2 were obtained by cloning the PCR products into pMD18-T after they were purified using agarose gel electrophoresis. Plasmid pET-NtrA and pET-arsC2 were obtained by inserting the \u003cem\u003eBsNtrA\u003c/em\u003e and \u003cem\u003earsC2\u003c/em\u003e genes into the pET-15b vector digested with \u003cem\u003eNde\u003c/em\u003e I and \u003cem\u003eXho\u003c/em\u003e I, respectively, following sequence verification of the recombinant pMD18-T plasmids. The recombinant plasmids were subsequently cultured after being converted into \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 (DE3) cells and designated names such as \u003cem\u003eE. coli\u003c/em\u003e DENA1 and DEAC3. When the optical density at 600 nm reached 0.6, 1.0 mM isopropyl-β-D-1-thiogalactopyranoside was administered to the cell to synthesize the His-tagged BsNtrA protein. For four to five hours, the culture was incubated at 28\u0026deg;C. As previously reported, the His-tagged BsNtrA was purified using a Ni-NTA-Sefinose Column (Sangon) and subsequently examined using 10% SDS-PAGE [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. ArsC2 was purified by using the same process.\u003c/p\u003e\n\u003ch3\u003eNitroreductase and arsenate reductase activity analysis\u003c/h3\u003e\n\u003cp\u003eThe capacity of BsNtrA for reduction of Rox(V) (Sigma-Aldrich, MO, USA) was determined by Nitroreductase Activity Assay Kit (ShanghaiFushengIndustrialCo., Ltd., China) in vitro. Briefly, Rox(V) was incubated at 37\u0026deg;C in the presence or absence of 50\u0026micro;L (1\u0026micro;M) BsNtrA in a reaction solution (1 \u0026micro;M FMN/FAD and 500 \u0026micro;M NAD(P)H in 20 mM Tris/HCl, pH 8, and 100 mM NaCl. 100\u0026micro;L horseradish peroxidase (HRP) was added to the mixture and incubated at 37℃ for 15 min in the dark. The absorbance of the mixture was measured at 540nm wavelength. 3-amino-4-hydroxybenzenearsonic acid (AHA) was used as a control (CAS: 2163-77-16).\u003c/p\u003e \u003cp\u003eThe capacity of ArsC2 for reduction of HAPA(V) (Hubei Dahao Technology Co., Ltd., China) was determined by the Arsenate Reductase Activity Assay Kit (Shanghai Bohu Biological Technology Co., Ltd., China) in vitro. Briefly, 100\u0026micro;l (1\u0026micro;M) HAPA(V) was added into 50\u0026micro;l PBS buffer and incubated at 37℃ for 60 min after antibody addition. TMB substrate 100\u0026micro;l acted for 20 minutes at 37℃ in the dark. Difference values of absorbance (S) in the ArsC2 mixture and negative control (N) at 450nm (W1) and 630nm (W2) were measured. 1-Amino-2-hydroxybenzene (CAS: 95-55-6) was used as a control.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eBsNtrA and ArsC2 confer resistance to Rox(V) and HAPA(V) of M20\u003c/h2\u003e \u003cp\u003eTo investigate the Rox(V) resistance mechanism of the inorganic arsenic-resistant opportunistic pathogen \u003cem\u003eBrevundimonas\u003c/em\u003e sp. M20 genes on the M20 genome were analyzed. Downstream \u003cem\u003ears\u003c/em\u003e cluster [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] that contributes to arsenite methylation, three Rox(V) resistance genes were identified (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Genes of FKQ52_RS09665, FKQ52_RS14070 and FKQ52_RS14075, encoding nitroreductase, arsenate reductase and MFS transporter, were named \u003cem\u003eBsNtrA\u003c/em\u003e, \u003cem\u003earsC\u003c/em\u003e2 and \u003cem\u003eBsExpA\u003c/em\u003e respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The toxic trivalent amino aromatic derivative 4-hydroxy-3-aminophenylarsenite (HAPA(III)) was developed in \u003cem\u003eSinorhizobium meliloti\u003c/em\u003e by the sequential reduction of the arsenate group in Rox(V) after nitroreductase catalyzed the reduction of the nitro group to form an intermediate product 4-hydroxy-3-aminophenylarseate (HAPA(V)) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Thus, resistance to Rox(V) and HAPA(V) of M20 was tested. M20 showed resistance to both Rox(V) and HAPA(V), with MIC of 1.25 mM and 1.50 mM, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFunctions of BsNtrA, ArsC2 and BsExpA in M20 were analyzed by gene expression via pMD18-T. MICs of \u003cem\u003eE. coli\u003c/em\u003e EcNAE3 (containing \u003cem\u003eBsNtrA\u003c/em\u003e, \u003cem\u003earsC\u003c/em\u003e2 and \u003cem\u003eBsExpA\u003c/em\u003e), EcN1(containing \u003cem\u003eBsNtrA\u003c/em\u003e), EcC1(containing \u003cem\u003earsC\u003c/em\u003e2), EcE1 (containing \u003cem\u003eBsExpA\u003c/em\u003e), EcNC (containing \u003cem\u003eBsNtrA\u003c/em\u003e and \u003cem\u003earsC\u003c/em\u003e2), EcNE (containing \u003cem\u003eBsNtrA\u003c/em\u003e and \u003cem\u003eBsExpA\u003c/em\u003e), and EcCE (containing \u003cem\u003earsC\u003c/em\u003e2 and \u003cem\u003eBsExpA\u003c/em\u003e) to Rox(V) and HAPA(V) were tested. The MIC of Rox(V) against \u003cem\u003eE. coli\u003c/em\u003e BsNAE3 was raised to 1.875 mM compared to DH5α and DT. When incubated in the presence of HAPA(V), \u003cem\u003eE. coli\u003c/em\u003e BsNAE3 also emerged as a resistance, with a MIC of 1.75 mM. Thus, the expression of \u003cem\u003eBsNtrA\u003c/em\u003e, \u003cem\u003earsC\u003c/em\u003e2 and \u003cem\u003eBsExpA\u003c/em\u003e in \u003cem\u003eE. coli\u003c/em\u003e brought the Rox(V) and HAPA (V) resistance (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eWhen any two genes of \u003cem\u003eBsNtrA\u003c/em\u003e, \u003cem\u003earsC\u003c/em\u003e2 and \u003cem\u003eBsExpA\u003c/em\u003e were co-expressed in \u003cem\u003eE. coli\u003c/em\u003e, MICs to Rox(V) and HAPA (V) showed differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). For EcNC, with \u003cem\u003eBsNtrA\u003c/em\u003e and \u003cem\u003earsC\u003c/em\u003e2 co-expression, Rox(V) and HAPA (V) resistance increased slightly, compared to DH5α and DT. Meanwhile, MICs to Rox(V) and HAPA (V) were 0.75 mM and showed no significant differences. \u003cem\u003eBsNtrA\u003c/em\u003e and \u003cem\u003eBsExpA\u003c/em\u003e co-expression did not offer Rox(V) and HAPA (V) resistance, compared to DH5α and DT. Meanwhile, resistance to Rox(V) and HAPA (V) exhibited significant differences when \u003cem\u003earsC\u003c/em\u003e2 and \u003cem\u003eBsExpA\u003c/em\u003e were co-expressed. A slight increase of Rox(V) resistance (MIC\u0026thinsp;=\u0026thinsp;0.25 mM) and a significant increase of HAPA (V) resistance (MIC\u0026thinsp;=\u0026thinsp;3.75 mM) were observed.\u003c/p\u003e\u003cp\u003eSingle gene expression in \u003cem\u003eE. coli\u003c/em\u003e showed no changes of MICs to Rox(V) and HAPA (V) in EcN1 and EcE1. MIC of Rox(V) and HAPA (V) toward EcC1, with \u003cem\u003earsC\u003c/em\u003e2 expression, showed a similar increase when \u003cem\u003earsC\u003c/em\u003e2 and \u003cem\u003eBsExpA\u003c/em\u003e were co-expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Thus, the significant rise of HAPA (V) resistance in \u003cem\u003eE. coli\u003c/em\u003e DH5α was attributed to the \u003cem\u003earsC\u003c/em\u003e2 expression. Considering the substantial increase in Rox(V) resistance in \u003cem\u003eBsNtrA\u003c/em\u003e, \u003cem\u003earsC\u003c/em\u003e2 and \u003cem\u003eBsExpA\u003c/em\u003e co-expression strain EcNAE3 and protein functions, BsNtrA provided the catalysis of the nitro group in Rox(V). Accordingly, it was suggested that Rox(V) reduction in M20 is a two-step simultaneous decrease of the nitro group and arsenic atom, similar to that found in \u003cem\u003eS. meliloti\u003c/em\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBsNtrA FMN interaction pocket\u003c/h3\u003e\n\u003cp\u003eThe protein structure of BsNtrA was modeled using homology to investigate its function further. BsNtrA from \u003cem\u003eBrevundimonas sp\u003c/em\u003e. M20 exhibited dimer formation, aligning with the homodimeric nature common to most nitroreductases [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To form a globular structure with expanded areas at both termini, two monomers of BsNtrA combine to form an α\u0026thinsp;+\u0026thinsp;β fold domain. With five β-strands and seven α-helices, each monomeric BsNtrA comprises three structural segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The FMN binding motif is characterized by a conserved sequence, \"RRS-PDH-WEW-W\u0026times;T/S\" (Figure S2). Two FMN molecules could interact with both monomers in this homodimer by binding in the deep pockets of the dimeric interface. They have an opening to the surface and are attached to the pocket at the dimeric interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The coenzyme FMN's isoalloxazine ring is orientated with its isoalloxazine ring packed against the β3 backbone atoms. It extends into the deep pockets created by the other dimeric subunit's Trp131 residue, the α5-helix, and the α2\u0026ndash;β1 loop from one of the monomers. P57-H57 and W148-T150 mediate the entrance of the isoalloxazine ring into the pocket. R26-S28 and R187-R188 mediate interactions with the ribityl moiety of FMN (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBsNtrA catalyzed the nitro group reduction of Rox(V)\u003c/h2\u003e \u003cp\u003eFor Rox(V) nitroreduction, the open FMN cofactor-binding pocket of BsNtrA also provides a reaction zone. Residues H57, G58, K59 and FMN mediate interactions within 5.0 \u0026Aring; along the nitro of Rox(V). The imidazole group of His57, acylamino of Gly58 and amidogen of Lys 58 could interact with O6 of the nitro moiety in ROX(V) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Meanwhile, the open reaction pocket facilitates the entry and exit of substrates and products (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). A Nitroreductase Activity Assay Kit was used to assess the nitroreductase activity of BsNtrA in the presence of NAD(P)H and FMN. A combination of Rox(V) and BsNtrA was active. At 450 nm, however, the AHA and BsNtrA mixture displayed a decrease in absorbance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Considering the difference between Rox(V) and AHA, a nitro group, this result confirmed that BsNtrA can reduce nitro-containing Rox(V).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eArsC2 has HAPA(V) reduction catalytic activity\u003c/h2\u003e \u003cp\u003eInorganic arsenate must first be reduced to arsenite by arsenate reductase (ArsC) before it is extruded [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. \u003cem\u003earsNRBC\u003c/em\u003e operon was identified in previous research in the M20 chromosome (unpublished). Another arsenate reductase encoded by \u003cem\u003eRS14070\u003c/em\u003e and named ArsC2 in the M20 chromosome was determined. As an organic arsenic compound, Rox(V) is not a suitable substrate for ArsC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Surprisingly, HAPA(V) reduced activated the arsenate reductase enzyme activity of ArsC2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). As an analogue of HAPA(V), 1-Amino-2-hydroxybenzene (AHB) could not activate the arsenate enzyme activity of ArsC2 because of the AsO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e deficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Thus, these results showed a reduction of HAPA(V) to HAPA(III) mediated by ArsC2.\u003c/p\u003e \u003cp\u003eTo investigate the detoxification mechanism, we analyzed the details of ArsC2 and its catalytic function. It indicated that ArsC2 mutated into a unique protein in M20. As reductases, essential redox-active cysteines are catalytic residues in ArsC, but the cysteine numbers vary. ArsC contains three cysteines (Cys10, Cys82 and Cys89) in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and \u003cem\u003eBacillus thuringiensis\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], two cysteines in \u003cem\u003eE. coli\u003c/em\u003e (Cys12 and Cys106) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and two cysteines in M20 (Cys11 and Cys105). Unlike these arsenate reductases, ArsC2 only contains one cysteine (Cys11, equal to Cys12 in \u003cem\u003eE. coli\u003c/em\u003e), the catalytic residue [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. ArsC2 maintained the HX3CX3R type catalytic sequence motif, which is necessary for arsenic detoxification activity in the traditional arsenate-reductase family, in addition to Cys11, His7, and Arg15 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] (Fig S3). This conservative motif provided a catalytic ability to pentavalent arsenic.\u003c/p\u003e \u003cp\u003eLike ArsC in \u003cem\u003eE. coli\u003c/em\u003e (PDB: 1I9D), ArsC2 of M20 comprised four-stranded β-sheets and seven α-helices. The difference between them is the composition and structure of random coils (RC). Catalytic residue at the end of α1 was sounded by three random coils (RC1-3) to form an active site loop, and compared to 1I9D, RC1-3 of ArsC2 provided a larger substrate binding space (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The amino hydroxybenzene tail of HAPA(V) is embedded into this loop and stabilized by residues, such as Thr13, Val17, Arg93, Val103, Leu104, Ala105 and Arg106 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Moreover, RC1-3 offered a higher hydrophobic environment-sounding active site, especially the mutation of Cys106 in 1I9D to Ala105 in ArsC2. This increased hydrophobic environment created a hydrophobic trap to enter HAPA's amino hydroxybenzene tail (V). Besides the hydrophobic trap, substrate binding space changed from a canyon-like type (11.477\u0026times;9.826 \u0026Aring;) to a basin-like type (12.264 \u0026times;18.380 \u0026Aring;) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). This expanded area, combined with the hydrophobic trap, may be a reasons for substrate specificity changes from inorganic arsenate to organic arsenic.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBsexpA has HAPA(III) efflux pump function\u003c/h2\u003e \u003cp\u003eConsidering that expression of \u003cem\u003eBsexpA\u003c/em\u003e alone in \u003cem\u003eE. coli\u003c/em\u003e did not increase the MIC to Rox(V) and HAPA(V) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), we concluded that BsexpA is a HAPA(III) specific efflux pump and investigated the transport mechanism and cure for Rox(V) resistance.\u003c/p\u003e \u003cp\u003eBsexpA is a \u003cem\u003eBrevundimonas\u003c/em\u003e sp. specific MFS family transporter. Rosetta Design's homologous modeling revealed that the structure of BsexpA comprises two domains (the C domain and the N domain) and 12 TM helices (TMs 1\u0026ndash;12). The interaction of BsexpA and HAPA(III) analysis indicated two substrate binding sites (SBS1 and SBS2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In SBS1, N32, A33, I36, and D37, residues bind mainly to HAPA(III) with carbon-hydrogen bond, Pi-alkyl and unfavorable acceptor-acceptor type acting forces. In SBS2, interacting residues moved to R127, V128, S354 and G358 with attractive charge and π bond forces (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), which are weaker than SBS1. These two binding sites indicated a substrate delivery between them when conformations change outward-open and inward-open in typical MFS [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePotential flavonoid EPI baicalin (CAS: 21967-41-9) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) has been investigated previously [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The inhibition of baicalin towards BsexpA was then evaluated using molecular simulation. The baicalin binding site (BBS) was in the middle cavity between SBS1 and SBS2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC up). Nine residues, including HAPA(III) binding residue R127 in SBS1, interacted with baicalin by more solid bonds such as van der Waals, salt bridge, Pi-anion and carbon-hydrogen bond (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC down). This binding characteristic of baicalin with BsexpA provided two effects on HAPA (III) transport. Firstly, stronger interaction of BsExpA with baicalin than with HAPA(III) may make it easier to bind with baicalin. Secondly, once baicalin binds to BsExpA, BBS occupies the HAPA (III) delivery space from SBS1 to SBS2 by steric hindrance and grabs the R127 residues to prevent HAPA (III) binding to SBS2. Thus, these characteristics may prevent HAPA (III) transportation to the extracellular. Effects of baicalin on BsexpA were investigated. For M20 and EcE1, baicalin decreased MIC towards HAPA(III) to \u0026lt;\u0026thinsp;0.25 mM both and showed no influences on Rox(v) and HAPA(V) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These above strains show resistance to baicalin (MIC\u0026thinsp;\u0026gt;\u0026thinsp;8 mM). This result is consistent with a previous report that the MIC of baicalin to \u003cem\u003eE. coli\u003c/em\u003e strains was 8.96 mM [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe contamination of organoarsenic is becoming increasingly prominent [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Roxarsone is an organoarsenical compound widely used for decades and is a significant threat to the eco-environment and human health [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. It has been demonstrated that microbial communities are crucial to the worldwide biocycles of arsenic [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. \u003cem\u003eBrevundimonas\u003c/em\u003e sp. M20, isolated previously, is resistant to inorganic arsenic and Rox(V). \u003cem\u003earsHRNBC\u003c/em\u003e cluster in the M20 genome is responsible for inorganic arsenic resistance. This study demonstrates that an FMN-dependent nitroreductase BsntrA and an arsenate reductase ArsC2 provide the resistance to Rox(V). These two protein is responsible for the Rox(V) nitro reduction and AS(V) reduction, respectively. These findings reveal a novel mechanism that provides defense against environmental aromatic arsenicals.\u003c/p\u003e \u003cp\u003eThe reduction of nitro groups in nitroaromatic and nitro heterocyclic compounds to amino or hydroxylamino groups is mediated by the enzyme nitroreductase, which depends on FMN[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The nitroreductase family has been identified in eukaryotic, archaeal, and bacterial species [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Nitroreductases with broad substrate specificity can catalyze both natural and produced molecules. As a result, the biotechnological uses of nitroreductases in prodrug activation gene therapy for the treatment of cancer, bioremediation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and biocatalysis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] make them extremely attractive [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. PpNfnB from \u003cem\u003eP. putida\u003c/em\u003e catalyzes FMN-NADPH-dependent nitroreduction of Rox(III) to less toxic HAPA(III) and confers resistance to Rox(V) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. MdaB from \u003cem\u003eSinorhizobium meliloti\u003c/em\u003e both reduces MAs(V) to MAs(III) and catalyzes sequential two-step reduction of nitro and arsenate groups in Rox(V), producing the highly toxic trivalent amino aromatic derivative HAPA(V) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Crystal structures of nitroreductases, such as YdjA [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and NfsA [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] in \u003cem\u003eE. Coli\u003c/em\u003e, Frm2 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] from \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e were elucidated. Nitroreductase of M20, BsNtrA, formed a dimer by seven α-helices and five β-strands, consistent with the homodimer characteristic of most nitroreductases [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The activity confirmed that BsNtrA can reduce nitro-containing Rox(V). This nitroreductase provides the nitro reduction of Rox(V). Thus, BsNtrA is a promising enzyme for bioremediation.\u003c/p\u003e \u003cp\u003eArsenate reductase (ArsC) facilitates the two-electron reduction of arsenate to arsenite, serving as the essential enzyme in the arsenate reduction process [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. \u003cem\u003earsC\u003c/em\u003e is usually located in \u003cem\u003ears\u003c/em\u003e operons in various microorganisms [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. ArsC contains three cysteines (Cys10, Cys82 and Cys89) in \u003cem\u003eStaphylococcus aureus\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and \u003cem\u003eBacillus thuringiensis\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], two cysteines in \u003cem\u003eE. coli\u003c/em\u003e (Cys12 and Cys106) and two cysteines in M20 (Cys11 and Cys105). Cys12 was identified as the catalytic residue and is activated by a nearby positive charge, His-8, in \u003cem\u003eE. coli\u003c/em\u003e ArsC [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, Besides ArsC encoded by \u003cem\u003earsC\u003c/em\u003e gene in an \u003cem\u003ears\u003c/em\u003e operon, M20 contains a novel arsenate reductase encoding gene (\u003cem\u003earsC\u003c/em\u003e2). The organic arsenic compound HAPA(V), reduced from Rox(v), is not a suitable substrate for ArsC but activates the arsenate reductase enzyme activity of ArsC2. Thus, these results indicated a reduction of the HAPA(V) arsenate group mediated by ArsC2. Moreover, unlike two or more cysteines containing ArsCs, ArsC2 is a one-Cys containing (Cys11) arsenate reductase. The classical arsenate-reductase family's arsenic detoxification activity is contingent upon the remaining cysteines in the HX\u003csub\u003e3\u003c/sub\u003eCX\u003csub\u003e3\u003c/sub\u003eR-type catalytic sequence motif [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Compared to 1I9D from \u003cem\u003eE. coli\u003c/em\u003e [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], mutations of residues that sound active site cavities not only provided a larger substrate binding space for HAPA(V) binding but also increased hydrophobic environment to create a hydrophobic trap to facilitate the higher hydrophobic substrate entry. These results may explain why ArsC2 could catalyze HAPA(V).\u003c/p\u003e \u003cp\u003eArsenite transporters enable various organisms to flourish in arsenic-contaminated environments despite the arsenate reductase. In this stage, an arsenite transporter catalyzes an energy-dependent process that pumps the arsenite out of the cell's interior or stores it in vacuoles [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. To date, five types of arsenic transporters (ArsB [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], Acr3[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], ArsJ [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], ArsP [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e], ArsK [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and MSF1 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]) have been found in various arsenic-resistant bacteria, and each confers resistance to different types of arsenic compounds [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. ArsJ, ArsK and MSF1 belong to the major facilitator superfamily (MFS) and mediate the transport of various substrates [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Adjacent to the \u003cem\u003earsC\u003c/em\u003e2 gene, a MFS transporter encoding gene \u003cem\u003eBsExpA\u003c/em\u003e was identified. Gene expression showed that this gene contributes to resistance to Rox(V) and HAPA(V), partly only co-expressed with \u003cem\u003earsC2\u003c/em\u003e. Thus, the function of BsExpA is related to the action of ArsC2. The deductive substrate is HAPA(V).\u003c/p\u003e \u003cp\u003eThe rapid emergence of antimicrobial resistance has become a serious clinical challenge. The therapeutic effectiveness of antibiotics is improved by identifying efficient EPIs that prevent bacteria from pumping antibiotics out of the cell [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Verapamil, reserpine, carbonyl cyanide-m-chlorophenylhydrazone, and β-naphthylamide have all been shown to be EPIs. However, most of these compounds' clinical uses were restricted by their toxicity, volatility, and low bioavailability; as a result, separating EPIs from natural products\u0026mdash;such as flavonoids\u0026mdash;has been a successful technique [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Luteolin [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], 5'-Methoxyhydnocarpin-D [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and 4\u0026prime;, 6\u0026prime; -dihydroxy-3\u0026prime;, 5\u0026prime;-dimethyl-2\u0026prime; -methoxychalcon[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] were found to be EPIs in previous studies. As a flavonoid compound, baicalin inhibited MFS-type efflux pump BsExpA in this study. Unlike preventing the efflux pump from obtaining energy (such as luteolin towards MsrA[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]), baicalin binds to BsExpA by more solid bonds, compared to the bonds of substrate binding. Moreover, the baicalin binding site in the middle cavity between HAPA(III) binding sites grabbed R127, an essential HAPA(III) binding residue. Thus, these characteristics may offer baicalin a stronger interaction with BsExpA than HAPA(III) and a steric hindrance to prevent HAPA (III) transportation to the extracellular.\u003c/p\u003e \u003cp\u003eThe data presented herein indicates a contribution to Rox(V) resistance mediated by nitroreductase, arsenate reductase and MFS transporter in \u003cem\u003eBrevundimonas sp\u003c/em\u003e. M20 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This pathway reduces organic Rox(V) to HAPA(III) by a two-step reduction catalysis and pump-out. Firstly, nitroreductase BsNtrA catalyzes Rox(V) to HAPA(V) by reducing the nitro group into the amino group. Then, AsO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e in HAPA(V) was reduced to AsO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e by arsenate reductase ArsC2 to form HAPA(III). Thirdly, HAPA(III) was pumped out by MFS family transporter BsExpA. Elucidation of this novel Rox(V) resistance pathway adds an essential inspection to continue the arsenic resistance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe complete sequence of the \u003cem\u003eBrevundimonas\u003c/em\u003e sp. M20 genome was deposited in GenBank under accession no. CP041243.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the language of a draft of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National College Students\u0026rsquo; innovation and entrepreneurship training program [grant number S202410439004, 2024]. We thank Liwen Bianji (Edanz) (www.liwenbianji.cn) for editing the language of a draft of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXHZ prepared the strain construction, the data analysis and wrote the manuscript. MHY the performance of bioinformatics analysis. JHW helped to analyze data. CCL helped to analyze data and revised the manuscript. BW operated the determination of organoarsenical compounds reduction. YAW led the project and revised the manuscript. All authors read and approved the final manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYang HC, Rosen BP: New mechanisms of bacterial arsenic resistance. \u003cem\u003eBiomed J \u003c/em\u003e2016, 39(1):5-13.\u003c/li\u003e\n\u003cli\u003eChen J, Rosen BP: The Pseudomonas putida NfnB nitroreductase confers resistance to roxarsone. \u003cem\u003eSci Total Environ \u003c/em\u003e2020, 748:141339.\u003c/li\u003e\n\u003cli\u003eHan J-C, Zhang F, Cheng L, Mu Y, Liu D-F, Li W-W, Yu H-Q: Rapid Release of Arsenite from Roxarsone Bioreduction by Exoelectrogenic Bacteria. \u003cem\u003eEnvironmental Science \u0026amp; Technology Letters \u003c/em\u003e2017, 4(8):350-355.\u003c/li\u003e\n\u003cli\u003eTang R, Yuan Sj, Yulan Wang, Wei Wang, Hu Z-h: Arsenic volatilization in roxarsone-loaded digester: Insight into the main factors and arsM genes. \u003cem\u003eSci Total Environ \u003c/em\u003e2020, 711:135123.\u003c/li\u003e\n\u003cli\u003eCortinas I, Field JA, Kopplin M, John R Garbarino JR, Gandolfi J, Sierra-Alvarez R: Anaerobic biotransformation of roxarsone and related N-substituted phenylarsonic acids. \u003cem\u003eEnviron Sci Technol \u003c/em\u003e2006, 40(9):2951-2957.\u003c/li\u003e\n\u003cli\u003eStolz JF, Perera E, Kilonzo B, Kail B, Crable B, Fisher E, Ranganathan M, Wormer L, Basu P: Biotransformation of 3-nitro-4-hydroxybenzene arsonic acid (roxarsone) and release of inorganic arsenic by \u003cem\u003eClostridium \u003c/em\u003especies. \u003cem\u003eEnviron Sci Technol \u003c/em\u003e2007, 41(3):818-823.\u003c/li\u003e\n\u003cli\u003eYang Z, Peng H, Lu X, Liu Q, Huang R, Hu B, Kachanoski G, Zuidhof MJ, Le XC: Arsenic metabolites, including N-Acetyl-4-hydroxy-m-arsanilic acid, in chicken litter from a roxarsone-feeding study involving 1600 chickens. \u003cem\u003eEnviron Sci Technol \u003c/em\u003e2016, 50(13):6737-6743.\u003c/li\u003e\n\u003cli\u003eYao L, Careya MP, Zhong J, Bai C, Zhou C, Meharg AA: Soil attribute regulates assimilation of roxarsone metabolites by rice (\u003cem\u003eOryza sativa\u003c/em\u003e L.). \u003cem\u003eEcotoxicol Environ Saf \u003c/em\u003e2019, 184:109660.\u003c/li\u003e\n\u003cli\u003eFrensemeier LM, B\u0026uuml;ter L, Vogel M, Karst U: Investigation of the oxidative transformation of roxarsone by electrochemistry coupled to hydrophilic interaction liquid chromatography/mass spectrometry. \u003cem\u003eJournal of Analytical Atomic Spectrometry \u003c/em\u003e2017, 32(1):153-161.\u003c/li\u003e\n\u003cli\u003eYan Y, Chen J, Galván AE, Garbinski LD, Zhu Y-G, Rosen BP, Yoshinaga M: Reduction of organoarsenical herbicides and antimicrobial growth promoters by the legume \u003cem\u003eSymbiont Sinorhizobium meliloti\u003c/em\u003e. \u003cem\u003eEnviron Sci Technol \u003c/em\u003e2019, 53(23):13648-13656.\u003c/li\u003e\n\u003cli\u003eKim SG, Chung J-S, Sutton B, Lee J-S, L\u0026oacute;pez-Maury L, Lee SY, Florencio FJ, Lin T, Zabet-Moghaddam M, Wood MJ\u003cem\u003e et al\u003c/em\u003e: Redox, mutagenic and structural studies of the glutaredoxin/arsenate reductase couple from the cyanobacterium \u003cem\u003eSynechocystis \u003c/em\u003esp. 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novel mechanism of action of ketoconazole: inhibition of the \u003cem\u003eNorA \u003c/em\u003eefflux pump system and biofilm formation in multidrug-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. \u003cem\u003eInfect Drug Resist \u003c/em\u003e2019, 12:1703-1718.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Roxarsone (V), Nitroreductase, Arsenate reductase, MFS efflux pump, Baicalin, Efflux pump inhibitor","lastPublishedDoi":"10.21203/rs.3.rs-5363972/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5363972/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRoxarsone (V) (Rox(V)) is an organoarsenical compound that poses significant risks to aquatic ecosystems and contributes to various diseases through its conversion into mobile inorganic and more toxic arsenic. Reducing trivalent 3-amino-4-hydroxyphenylarsonic acid (HAPA(III)) offers a competitive advantage; however, it leads to localized arsenic contamination, which can disrupt the soil microbiome and impede plant growth. Three genes, \u003cem\u003eBsntrA\u003c/em\u003e, \u003cem\u003earsC\u003c/em\u003e2, and \u003cem\u003eBsexpA\u003c/em\u003e, encoding nitroreductase, arsenate reductase, and MFS transporter, were identified in a Rox(V) resistant strain \u003cem\u003eBrevundimonas\u003c/em\u003e sp. M20. Then, a three-step approach, including nitroreduction, As (V) reduction, and HAPA(III) secretion, which is responsible for Roxarsone(V) resistance, was confirmed. Moreover, the flavonoid compound baicalin occupies the HAPA (III) delivery space and grabs the R127 residues by stronger interaction and steric hindrance to prevent HAPA (III) transported by BsexpA to the extracellular. These results demonstrate a new Rox(V) reduction pathway, providing a potential efflux pump inhibitor to trap higher toxins.\u003c/p\u003e","manuscriptTitle":"A reduction-secretion system contributes to roxarsone (V) degradation and efflux in Brevundimonas sp. 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